The present Invention relates to a novel serine-threonine kinase gene.
Fetal tissues are comprised of many undifferentiated cells that proliferate actively, highly activated cells, nascent vascular endothelial cells, and so on. Although the activity of these cells in fetal tissues is stringently regulated and inhibited as individuals mature, the state of fetal tissues can be considered similar to that of a solid tumor except that the activity is regulated. Therefore, some of the genes expressed specifically or more strongly in fetal tissues (fetal genes) can be genes involved in the phenomena characteristic of solid tumors such as abnormal growth, immortalization, infiltration, metastasis, and angiogenesis. In addition, some diseases other than tumors are also supposed to arise because fetal genes, which are repressed in a normal living body, are abnormally activated. Therefore, genes involved in various diseases such as tumors can be screened by isolating and analyzing fetal genes.
However, there are still few reports on systematic analysis focusing merely on fetal genes from these viewpoints, and at present there is a far from perfect understanding of these gene groups.
An objective of this invention is to isolate genes expressed specifically in fetal tissues and to screen genes related to diseases.
The present inventors thought that fetal tissue cells could be a model for solid tumor cells and that genes involved in diseases such as tumors could be screened by isolating and analyzing fetal genes. Furthermore, the present inventors thought it possible to develop a medicine with a novel action mechanism by designing drugs targeting the genes. Based on these thoughts, the present inventors have tried to isolate fetal genes.
Specifically, the present inventors prepared a subtraction library with many genes expressed specifically in fetal livers (or more strongly than adult livers) by the suppression subtractive hybridization method, extracted clones from this library at random, and analyzed their structure. By doing so, the present inventors succeeded in isolating a novel gene, VRK1, having the consensus sequence of a serine-threonine kinase active site. The present inventors also performed a data base search based on the amino acid sequence deduced from the isolated gene. The present inventors thus have found this gene product exhibits a significant homology with B1R kinase, which is presumably involved in DNA replication of vaccinia virus. In addition, the present inventors found human EST having a very high homology with this gene in the database and isolated its full-length cDNA, VRK2. Analyzing the expression of the two isolated genes in various cells by northern blot analysis showed that these genes are strongly expressed, especially in actively growing cells such as human fetal livers, testes, and various tumor cell lines. Furthermore, the present inventors have found that the VRK1 protein actually has protein kinase activity.
Thus, the present invention relates to novel serine-threonine kinase genes, VRK1 and VRK2. More specifically, the present invention relate to:
(1) a protein having the amino acid sequence of SEQ ID NO: 2, or a protein having the same amino acid sequence where one or more amino acids are added, deleted, or substituted and having serine-threonine kinase activity,
(2) a protein having the amino acid sequence of SEQ ID NO: 4, or a protein having the same amino acid sequence where one or more amino acids are added, deleted, or substituted and having serine-threonine kinase activity,
(3) a protein encoded by a DNA sequence that hybridizes with the DNA sequence of SEQ ID NO: 1 or its complementary sequence and having serine-threonine kinase activity,
(4) a protein encoded by a DNA sequence that hybridizes with the DNA sequence of SEQ ID NO: 3 or its complementary sequence and having serine-threonine kinase activity,
(5) a DNA encoding the protein of any one of (1) to (4),
(6) a vector comprising the DNA of (5),
(7) a transformant carrying the vector of (6),
(8) a method of producing the protein of any one of (1) to (4), wherein the method comprises cultivating the transformant of (7),
(9) an antibody binding to the protein of any one of (1) to (4),
(10) an antisense DNA against the DNA of (5) or part of it,
(11) a method of screening compounds having inhibitory activity of serine-threonine kinase activity of the protein of any one of (1) to (4), wherein the method is comprised of
(a) contacting the protein of any one of (1) to (4) with a substrate to be phosphorylated by this protein in the presence of a test compound to detect the kinase activity of the protein of any one of (1) to (4), and
(b) comparing the kinase activity detected in step (a) with that detected in the absence of the test compound and selecting a compound that lowers the kinase activity of the protein of any one of (1) to (4).
The present invention relates to novel serine-threonine kinases, xe2x80x9cVRK1xe2x80x9d and xe2x80x9cVRK2.xe2x80x9d The nucleotide sequence of the xe2x80x9cVRK1xe2x80x9d cDNA and the amino acid sequence of the protein are shown in SEQ ID NO: 1 and 2, respectively. In addition, the nucleotide sequence of the xe2x80x9cVRK2xe2x80x9d cDNA and the amino acid sequence of the protein are shown in SEQ ID NO: 3 and 4, respectively. xe2x80x9cVRK1xe2x80x9d cDNA has a significant homology with B1R kinase, which is presumably involved in DNA replication of vaccinia virus. The gene is also characterized by its strong expression in actively growing cells such as fetal livers, testes, and various tumor cell lines. In addition, overexpression of xe2x80x9cVRK1xe2x80x9d protein drastically increases the growing activity of NIH3T3 cells. These facts imply xe2x80x9cVRK1xe2x80x9d is involved in the regulation mechanism of cell growth. xe2x80x9cVRK1xe2x80x9d protein has protein kinase activity, which presumably plays an important roll in the regulation of cell growth. xe2x80x9cVRK2xe2x80x9d has a high homology with xe2x80x9cVRK1,xe2x80x9d especially in the serine-threonine kinase site. xe2x80x9cVRK2,xe2x80x9d like xe2x80x9cVRK1,xe2x80x9d has a significant homology with B1R kinase, and the gene is characterized by its strong expression in actively growing cells such as fetal livers, testes, and various tumor cell lines. These facts imply xe2x80x9cVRK2xe2x80x9d has the same function as that of xe2x80x9cVRK1.xe2x80x9d
xe2x80x9cVRK1xe2x80x9d and xe2x80x9cVRK2xe2x80x9d proteins can be prepared as recombinant proteins with recombinant DNA techniques or as natural proteins. The recombinant proteins can be prepared, for example, by cultivating cells transformed with the DNAs encoding these proteins, as will be described later. Natural proteins can be isolated from fetal livers, testes, or tumor cell strains such as HeLa S3, in which these proteins are highly expressed, by a method well-known to one skilled in the art, such as affinity chromatography with the antibodies of the present invention as described later. Either polyclonal or monoclonal antibodies can be used. The polyclonal antibodies can be prepared from, for example, serum from small animals such as rabbits immunized with these proteins by, for example, ammonium sulfate precipitation, protein A- or protein G-column chromatography, DEAE ion exchange chromatography, affinity chromatography using a column coupled with these proteins, etc. The monoclonal antibodies can be prepared as follows. First, a small animal such as a mouse is immunized with these proteins. The spleen is extracted from the mouse and dissociated to cells. The resulting cells are fused to mouse myeloma cells using a reagent such as polyethylene glycol, and the clone that produces antibodies against these proteins is screened from the fusion cells (hybridoma) thus generated. The hybridoma thus obtained is then transplanted into a mouse abdominal cavity. Ascites is collected from the mouse and purified by, for example, ammonium sulfate precipitation, protein A- or protein G-column chromatography, DEAE ion exchange chromatography, affinity chromatography using a column coupled with xe2x80x9cVRK1xe2x80x9d or xe2x80x9cVRK2xe2x80x9d protein, etc. If the antibodies obtained are to be used for administering to a human body (for antibody therapy or the like, etc.), humanized antibodies or human antibodies should be used to decrease immunogenicity. An example of methods for humanizing antibodies is the CDR graft method, in which an antibody gene is cloned from monoclonal antibody-producing cells and its antigenic determinant is transplanted to an existing human antibody. Besides, human antibodies can be directly prepared just like usual monoclonal antibodies by immunizing a mouse whose immune system is replaced with a human immune system.
Furthermore, one skilled in the art can prepare not only natural xe2x80x9cVRK1xe2x80x9d and xe2x80x9cVRK2xe2x80x9d proteins (SEQ ID NO: 2 and 4, respectively) but also proteins with substantially the same function as that of the natural proteins, if needed, by replacing amino acids in the proteins by a well-known method. Besides, mutations of amino acids in proteins can occur naturally. Thus, mutant proteins with serine-threonine kinase activity that are generated by introducing amino acid substitution, deletion, or addition into the natural proteins are also included in the proteins of the present invention. Methods for amino acid alteration, for example, a site-directed mutagenesis system using PCR (GIBCO-BRL, Gaithersburg, Maryland), the oligonucleotide-mediated site-directed mutagenesis method (Kramer, W. and Fritz, HJ (1987) Methods in Enzymol., 154: 350-367), and the Kunkel method (Methods Enzymol. 85, 2763-2766 (1988)), are well-known to one skilled in the art. Furthermore, usually ten or less, preferably six or less, and more preferably three or less amino acids are substituted. For example, proteins functionally equivalent to the VRK1 or VRK2 protein can be produced by conservative amino acid substitutions at one or more amino acid residues. A xe2x80x9cconservative amino acid substitutionxe2x80x9d is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The site where substitution, deletion, or addition is introduced is not particularly limited as long as the serine-threonine kinase activity is maintained. From the viewpoint of protein activity, the addition, deletion, or substitution of amino acids should be performed in a region other than the region corresponding to the consensus sequence of a serine-threonine kinase active site and to the consensus sequence of a protein kinase ATP binding site. Moreover, serine-threonine kinase activity of a protein can be detected, for example, by the method described in Example 9, mentioned later.
Furthermore, one skilled in the art can usually isolate DNAs having a high homology with the DNA encoding xe2x80x9cVRK1xe2x80x9d or xe2x80x9cVRK2xe2x80x9d protein SEQ ID NO: 1 or 3, respectively) based on the DNA or the part of it using a hybridization technique (Sambrook, J. et al., Molecular Cloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. Press, 1989) and obtain proteins having substantially the same function as VRK1 or VRK2 protein (SEQ ID NO: 2 or 4, respectively) from the DNA. Thus, proteins with serine-threonine kinase activity that are encoded by DNAs hybridizing with DNA encoding xe2x80x9cVRK1xe2x80x9d or xe2x80x9cVRK2xe2x80x9d protein are also included in the proteins of the present invention. Hybridizing DNAs are isolated from other organisms including, for example, mice, rats, rabbits, and bovines, and so on. Tissues such as fetal livers and testes are especially suitable for isolating. Thus isolated DNAs encoding proteins having substantially the same function as that of xe2x80x9cVRK1xe2x80x9d or xe2x80x9cVRK2xe2x80x9d proteins usually have a high homology with the DNA (SEQ ID NO: 1 or 3) encoding xe2x80x9cVRK1xe2x80x9d or xe2x80x9cVRK2xe2x80x9d protein, respectively. The term xe2x80x9chigh homologyxe2x80x9d used herein means at least 40% or more, preferably 60% or more, and more preferably 80% or more of sequence identity at the amino acid level. From the viewpoint of the protein activity, a high homology should be found in the regions corresponding to the consensus sequence of a serine-threonine kinase active site and to the consensus sequence of a protein kinase ATP binding site.
To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions (e.g., overlapping positions)xc3x97100). In one embodiment the two sequences are the same length.
To determine percent homology between two sequences, the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877 is used. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to a VRK1 or VRK2 protein molecules. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See www.ncbi.nlm.nih.gov.
Furthermore, the present invention relates to a DNA that specifically hybridizes under moderate or highly stringent conditions to a DNA encoding a protein of the present invention and comprises at least 15 nucleotide residues. The DNA can be used, for example, as a probe to detect or isolate a DNA encoding a protein of the present invention, or as a primer for PCR amplification. An example is DNA consisting of at least 15 nucleotides complementary to the nucleotide sequence of SEQ ID NO: 1 or NO: 3.
Standard hybridization conditions (e.g., moderate or highly stringent conditions) are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6, hereby incorporated by reference. Moderate hybridization conditions are defined as equivalent to hybridization in 2xc3x97 sodium chloride/sodium citrate (SSC) at 30xc2x0 C., followed by one or more washes in 1xc3x97SSC, 0.1% SDS at 50-60xc2x0 C. Highly stringent conditions are defined as equivalent to hybridization in 6xc3x97sodium chloride/sodium citrate (SSC) at 45xc2x0 C., followed by one or more washes in 0.2xc3x97SSC, 0.1% SDS at 50-65xc2x0 C.
Examples of conditions for hybridization to isolate these DNAs are as follows. After prehybridization at 55xc2x0 C. for 30 minutes or longer, hybridization is performed by adding labeled probes and incubating at 37xc2x0 C. to 55xc2x0 C. for an hour or longer using xe2x80x9cExpressHyb Hybridization Solutionxe2x80x9d (CLONTECH). After that, the resulting hybridized products are washed three times for 20 minutes each at room temperature in 2xc3x97SSC and 0.1% SDS then once at 37xc2x0 C. in 1xc3x97SSC and 0.1% SDS. More preferably, after prehybridization at 60xc2x0 C. for 30 minutes or longer, hybridization is performed by adding labeled probes and incubating at 60xc2x0 C. for an hour or longer using xe2x80x9cExpressHyb Hybridization Solutionxe2x80x9d (CLONTECH). Thereafter, the hybridized products are washed three times for 20 minutes each at room temperature in 2xc3x97SSC and 0.1% SDS then twice at 50xc2x0 C. in 1xc3x97SSC and 0.1% SDS. Still more preferably, after prehybridization at 60xc2x0 C. for 30 minutes or longer, hybridization is performed by adding labeled probes and incubating at 68xc2x0 C. for an hour or longer using xe2x80x9cExpressHyb Hybridization Solutionxe2x80x9d (CLONTECH). Thereafter, the hybridized product is are washed three times for 20 minutes each at room temperature in 2xc3x97SSC and 0.1% SDS then twice at 50xc2x0 C. in 0.1xc3x97SSC and 0.1% SDS.
The present invention also relates to the DNAs encoding the above-described proteins of the present invention. The DNAs of the present invention include cDNAs, genomic DNAs, and synthetic DNAs as long as they encode the proteins of the present invention. The DNAs of the present invention can be used to produce the recombinant proteins. Specifically, the recombinant proteins can be prepared by inserting the DNA (for example, the DNA of SEQ ID NO: 1 or 3) of the present invention into a suitable expression vector, cultivating the transformant obtained by introducing the vector into suitable cells, and purifying the expressed proteins. For example, mammalian cells such as COS, CHO, or NIH3T3 cells; insect cells such as Sf9 cells; yeast cells; and E. coli cells can be used for producing the recombinant proteins. Vectors for expressing recombinant proteins in these cells vary depending on the host cells. For example, pcDNA3 (Invitrogen) or pEF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322) is used for mammalian cells; xe2x80x9cBAC-to-BAC baculovirus expression systemxe2x80x9d (GIBCO BRL), for insect cells; xe2x80x9cPichia Expression Kitxe2x80x9d (Invitrogen), for yeast cells; and pGEX-5X-1 (Pharmacia) or xe2x80x9cQIAexpress systemxe2x80x9d (Qiagen), for E. coli cells. Vectors can be introduced into host cells by, for example, the method using calcium phosphate, DEAE dextran, or cationic liposome DOTAP (Boehringer Mannheim); electroporation; the calcium chloride method; etc. The recombinant proteins can be purified from the obtained transformants by the usual methods such as the method described in xe2x80x9cThe Qiaexpressionist handbook, Qiagen, Hilden, Germany.xe2x80x9d
Furthermore, the DNAs of the present invention can be used for gene therapy of diseases caused by mutations in genomic DNAs. In gene therapy, the DNAs of the present invention are administered to a living body inserted into adenovirus vectors (e.g., pAdexLcw), retrovirus vectors (e.g., pZIPneo) and so on. They can be administered by either ex vivo methods or in vivo methods.
Furthermore, since the proteins of the present invention are presumably involved in the regulation of cell growth, antisense DNAs against the DNAs of he present invention or part of them can be used as inhibitors for developing cell growth or as antitumor agents. The antisense DNAs are administered to a living body directly or in the form of the vectors into which they have been inserted. The antisense DNAs can be synthesized by methods well known to one skilled in the art.
The present invention also relates to a method of screening compounds having inhibitory activity of serine-threonine kinase activity of the proteins of the present invention. This screening method consists of two steps. First, the protein of the present invention is caused to contact a substrate to be phosphorylated by this protein in the presence of a test compound to detect the kinase activity of the protein of the present invention. Second, the kinase activity detected In step (a) is compared with that detected in the absence of the test compound, and a compound that lowers the kinase activity of the protein of the present invention is selected.
Test compounds used for this screening method are not particularly limited and are generally low-molecular-weight compounds, proteins (including the above-described antibodies of the present invention), peptides, etc. Test compounds are either artificially synthesized or natural. Substrates are, for example, casein, IkBxcex1 protein, etc. The kinase activity of the protein of the present invention can be detected, for example, by adding ATP having radioactively labeled phosphate to the reaction system containing the protein of the present invention and the substrate and measuring the radioactivity of the phosphate attached to the substrate. Specifically, the activity is detected by the method described in Example 9. The compounds thus isolated can be used as cell growth inhibitors or antitumor agents. Moreover, the present inventors learned that xe2x80x9cVRK1xe2x80x9d protein phosphorylates IkBxcex1 protein. IkBxcex1 is thought to be rapidly degraded when phosphorylated, thereby releasing and activating NF-kB bound thereto. In addition, NF-kB is well known as a central transcriptional regulator that causes widespread immune reactions and inflammation reactions. Therefore, compounds that inhibit the kinase activity of the proteins of the present invention can be used as antiphlogistics and immunosuppressants.